Staphylococcus pseudintermedius is a skin and mucous membrane commensal of dogs and is the Organism most frequently associated with atopic dermatitis and wound infections in dogs. It is also implicated in the pathogenesis of canine pyoderma. In the past, antimicrobial agents have been used to successfully control S. pseudintermedius infections. However, in recent years, there has been an increased prevalence of methicillin resistance and multidrug resistance among S. pseudintermedius isolates. This has made treatment of infection difficult due to the limited number of antimicrobial agents used in veterinary medicine. Therefore, there is an urgent need (or the development of alternative, treatment strategies to combat S. pseudintermedius infections.
Surface proteins in S. aureus harboring a C-terminal sorting signal with an LPXTG motif are covalently attached onto the peptidoglycan cell wall by the enzyme sortase A (SrtA) which is encoded by the srtA gene. This 618 bp gene encodes a protein of 206 amino acids with a molecular weight of 23.59 kDa. The enzyme is a cysteine transpeptidase that recognizes the conserved LPXTG motif in surface proteins and specifically cleaves between the threonine (T) and glycine (G) residues. The active site cysteine of SrtA forms an acyl enzyme intermediate that is relieved by the nucleophilic attack of the amino group of the pentaglycine cross bridge in peptidoglycan synthesis precursors. The N-terminus of the enzyme is located in the cytoplasm while the C-terminal enzymatic region is located across the plasma membrane (type II membrane topology). His120, Cys184 and Arg197 form the catalytic triad of the enzyme and are found to be conserved among sortases in S. aureus. Surface proteins attached to peptidoglycan precursors are then incorporated into the bacterial cell wall and displayed on the surface.
There are at least 17-21 surface proteins in S. aureus that harbor the LPXTG motif. Most of these proteins are involved in virulence and pathogenesis. SrtA is essential for the functional assembly of all surface proteins harboring an LPXTG motif, and therefore plays a crucial role in the pathogenesis of S. aureus infections.
S. aureus mutants that do not have a functional srtA gene fail to assemble surface proteins on to the peptidoglycan cell wall. Also, they are unable to form abscess lesions in organ tissues or cause lethal bacteremia in mice models of S. aureus infections. Therefore, inhibition of SrtA could result in defective display of all the proteins harboring the LPXTG motif and render the organism less virulent. SrtA inhibitors, therefore, may be useful as anti-infective agents particularly for methicillin and multidrug resistant organisms, by disrupting the pathogenesis of bacterial infections. The earliest described inhibitors of SrtA include methane-thiosulfonates such as MESET, (2-sulfonatoethyl) methane-thiosulfonate and p-hydroxymercuribenzoic acid. All these compounds interact with Cys184 and render the enzyme inactive. Other natural or chemical compounds have also been examined for their abilities to inhibit SrtA. Although identification of inhibitors for SrtA has been ongoing for several years, it has gained momentum in the last decade due to the advancements in genomics and proteomics.
However, SrtA in S. pseudintermedius has not been previously examined. To satisfy this need in the art, the present investigators identified, cloned and expressed the srtA gene of S. pseudintermedius in E. coli, and used molecular dynamic simulation to generate a model of S. pseudintermedius SrtA. After identifying the active site of S. pseudintermedius SrtA, scaffold hopping and molecular docking were combined to virtually screen certain drug databases [NCI (National Cancer Institute Open Database) and ZINC (a database of commercially available compounds for virtual screening maintained by the Department of Pharmaceutical Chemistry, University of California San Francisco, Genentech Hall, San Francisco, Calif.)] to identify candidate inhibitors of S. pseudintermedius SrtA. The compounds are hereafter referred to by the identifiers assigned in their database. The identified candidates are suitable for inhibition of S. pseudintermedius virulence.